# Patent

## Title
Methods and reagents for detecting endotoxin

## Abstract

A reagent containing a purified horseshoe crab Factor C, particularly a recombinantly produced Factor C, and a surfactant can be used in a sensitive, rapid, and reproducible assay to detect endotoxin.

## Background
This application claims the benefit of and incorporates by reference provisional application Ser. No. 60/310,125 filed Jun. 28, 2001 now abandoned.


FIELD OF THE INVENTION
The invention relates to reagents and methods for detecting endotoxin.
BACKGROUND OF THE INVENTION
Gram negative bacterial endotoxin is a widespread contaminant of a variety of materials, such as water, food, pharmaceutical products, and parenteral preparations. The most commonly used tests for endotoxin contamination employ amebocyte lysates derived from horseshoe crab hemolymph. As populations of these animals decreases, however, it becomes increasingly important to develop rapid and reliable methods for detecting endotoxin that do not rely on the availability of horseshoe crab hemolymph.
BRIEF SUMMARY OF THE INVENTION
The invention provides reagents and methods for detecting endotoxin. One embodiment of the invention is a reagent for detecting endotoxin, comprising a purified horseshoe crab Factor C protein and a surfactant. The surfactant can be an amphoteric surfactant represented by the following formulae: 




wherein R1 is an alkylene radical having from 1 to 4 carbon atoms; Y and Y′ are each (1) hydrogen, (2) lower alkyl, or (3) hydroxy lower alkyl; R2 and R3 are each (1) lower alkyl or (2) hydroxy lower alkyl; n is 0 or 1, when n is 0, R4 is alkyl containing from about 8 to about 18 carbon atoms; when n is 1, R4 is an alkylene radical having from 1 to about 6 carbon atoms; R5 is an alkyl containing from about 8 to about 18 carbon atoms; and M is hydrogen, sodium, potassium or ammonium. The surfactant can be an anionic surfactant represented by the following formulae:

(R6)n1-(Y)Ar(SO3M)n2  (E)

R5OSO3M  (F)

wherein R5, Y, and M have the same meaning as set forth above; R6 is an alkyl from 8 to 24 carbon atoms; n1 is an integer from 1 to 3; n2 is 1 or 2; and Ar is phenyl or naphthyl. The surfactant can be a cationic surfactant represented by the following formula: 




wherein R5, Y, and Y′ have the same meaning as set forth above. The surfactant can be a nonionic surfactant represented by the following formula:

R5R7R8N→O  (H)

wherein R5, Y, and Y′ have the same meaning as set forth above and R7 and R8 are each methyl or ethyl. The surfactant can be those nonionic surfactants selected from the group consisting of the condensation product of about 10 to 30 moles of ethylene oxide with the monoester of a hexahydric alcohol containing 6 carbon atoms with the ester group containing 10 to 20 carbon atoms.

Even another embodiment of the invention is a method of detecting endotoxin in a test sample. A test sample is contacted with (1) a reagent comprising (a) a purified horseshoe crab Factor C protein and (b) a surfactant to form a test sample-reagent mixture and (2) a Factor C substrate to form a contacted test sample. Cleavage of the Factor C substrate generates a detectable signal. The contacted test sample is assayed for the presence or absence of the detectable signal. An amount of the detectable signal that is increased relative to a control sample that does not contain endotoxin indicates a presence of endotoxin in the test sample.
A further embodiment of the invention is a method of detecting endotoxin in a test sample. A test sample is contacted with (1) a reagent comprising (a) a recombinant Carcinoscorpius rotundicauda Factor C protein and (b) a surfactant and (2) N-t-BOC-Asp(Obzl)-Pro-Arg-7-Amido-4-methyl coumarin to form a contacted test sample. The Factor C protein is made by the method of culturing a host cell comprising a vector encoding the Factor C protein in a supernatant under conditions such that the Factor C protein is expressed into the supernatant. The contacted test sample is assayed for the presence or absence of a fluorescent signal. An amount of the detectable signal that is increased relative to a control sample that does not contain endotoxin indicates a presence of endotoxin in the test sample.
Another embodiment of the invention is a method of detecting endotoxin in a test sample. A test sample is contacted with a reagent comprising a purified horseshoe crab Factor C protein and a surfactant as described above to form a test sample-reagent mixture. The test sample-reagent mixture is contacted with a Factor C substrate, wherein cleavage of the Factor C substrate generates a detectable signal. The contacted test sample-reagent mixture is assayed for the presence or absence of the detectable signal. An amount of the detectable signal that is increased relative to a control sample that does not contain endotoxin indicates a presence of endotoxin in the test sample.
Even another embodiment of the invention is a method of detecting endotoxin in a test sample. A test sample is contacted with a reagent comprising a recombinant Carcinoscorpius rotundicauda Factor C protein and a surfactant. The Factor C protein is made by the method of culturing a host cell comprising a vector encoding the Factor C protein in a supernatant under conditions such that the Factor C protein is expressed into the supernatant. The test sample-reagent mixture is contacted with N-t-BOC-Asp(Obzl)-Pro-Arg-7-Amido-4-methyl coumarin. The contacted test sample-regent mixture is assayed for the presence or absence of a fluorescent signal. An amount of the fluorescent signal that is increased relative to a control sample that does not contain endotoxin indicates a presence of endotoxin in the test sample.
Yet another embodiment of the invention is a kit for detecting endotoxin. The kit comprises a reagent that comprises (a) a purified horseshoe crab Factor C protein and (b) a surfactant as described above, together with instructions for a method of detecting endotoxin in a test sample. The method comprises the steps of (1) contacting a test sample with a reagent comprising (a) a purified horseshoe crab Factor C protein and (b) a surfactant as described above to form a test sample-reagent mixture; (2) contacting the test sample-reagent mixture with a Factor C substrate, wherein cleavage of the Factor C substrate generates a detectable signal; and (3) assaying the contacted test sample-reagent mixture for the presence or absence of the detectable signal, wherein an amount of the detectable signal that is increased relative to a control sample that does not contain endotoxin indicates a presence of endotoxin in the test sample.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Graph showing effect of Zwittergent 3-14 and Tween 20 on recombinant Factor C activity.
FIG. 2. Graph showing effect of Zwittergent 3-14 and Tween 80 on recombinant Factor C activity.
FIG. 3. Graph showing effect of Zwittergent 3-14 and Triton X-114 on recombinant Factor C activity.
FIG. 4. Plot showing endotoxin detection using a DPR (N-t-BOC-Asp(Obzl)-Pro-Arg-7-Amido-4-methyl)-coumarin substrate.
FIG. 5. Plot showing endotoxin detection using a VPR (N-t-BOC-Val-Pro-Arg-7-Amido-4-methyl)-coumarin substrate.
FIG. 6. Graph showing Limulus Factor C activity at different Zwittergent concentrations.
FIG. 7. Graph showing endotoxin sensitivity in the presence and absence of surfactant.
FIG. 8. Graph showing results of a one hour, one-step endotoxin assay, using DPR-coumarin as a substrate.
FIG. 9. Graph showing effect of Zwittergent 3-14 and octylthiolglycoside on recombinant Factor C activity.
FIG. 10. Graph showing effect of Zwittergent 3-14 and Genapol C-100 on recombinant Factor C activity.
FIG. 11. Graph showing effect of Zwittergent 3-14 and TX-100 on recombinant Factor C activity.



DETAILED DESCRIPTION OF THE INVENTION
The invention is a reagent for detecting endotoxin and a method of using the reagent. The reagent comprises a purified horseshoe crab Factor C protein and a surfactant. This reagent can be used in conjunction with a substrate for Factor C that, upon cleavage, generates a detectable signal to detect endotoxin in a test sample. The presence of the surfactant enhances the activation of the purified Factor C by endotoxin by as much as 3-7-fold, permitting more rapid and sensitive measurement of endotoxin levels in a test sample. The reagent preferably contains a recombinant Factor C, thus eliminating the need for a continuous supply of horseshoe crab hemolymph.
Purified Factor C
One component of the reagent of this invention comprises a purified horseshoe crab Factor C protein. Purified native Factor C from any of the four known horseshoe crab species, Limulus polyphemus, Carcinoscorpius rotundicauda, Tachypleudus tridentata, or Tachypleudus gigas, can be used in the practice of the invention. The native Factor C can be purified biochemically or purified Factor C can be produced recombinantly.
“Purified Factor C” as used herein means a composition of a Factor C protein as defined hereinafter that contains less than 30% by weight of non-Factor C native amebocyte lysate components from any of the four known horseshoe crab species. The composition specifically includes a cell culture supernatant which comprises recombinant Factor C protein (see below). Methods for purifying horseshoe crab Factor C from its native source are known and are disclosed, for example, in Nakamura et al., Eur. J Biochem. 154, 511-21, 1986; Navas et al., Biochem. Intl. 21, 805-13, 1990; Tokunaga et al., J. Biochem. 109, 150-157, 1991; U.S. Pat. Nos. 5,985,590, and 5,716,834. Any of these methods or their equivalents can be used to obtain a purified Factor C. See also Example 11. Purity of a Factor C preparation can be assessed by any means known in the art, such as SDS gel electrophoresis.
Preferably, the purified Factor C is recombinantly produced. Amino acid sequences for native Factor C from Tachypleus tridentata and Carcinoscorpius rotundicauda are known, as are the naturally occurring coding sequences for these proteins. SEQ ID NOS:1 and 3 provide coding sequences for the Tachypleus tridentata Factor C amino acid sequences shown in SEQ ID NOS:2 and 4, respectively. SEQ ID NOS:5 and 7 provide coding sequences for the Carcinoscorpius rotundicauda Factor C amino acid sequences shown in SEQ ID NOS:6 and 8, respectively. Because of the degeneracy of the genetic code, many other sequences can be envisioned that will encode each of these native Factor C proteins, and the invention specifically contemplates use of any of these coding sequences to produce a purified Factor C.
The invention also encompasses use of purified naturally and non-naturally occurring, i.e., recombinantly produced, Factor C variants, provided that the variants retain a Factor C enzyme activity. Factor C enzyme activity can be assessed using any assay for Factor C enzyme activity known in the art. See, e.g., Tokunaga et al., J. Biochem. 109, 150-57 (1991) and Nakamura et al., Eur. J. Biochem. 176, 89-94, 1988. The endotoxin assays described in Examples 1 and 13, below, also can be used. Naturally occurring Factor C variants include, for example, products of Factor C mRNA splice variants or mutated Factor C genes.
Non-naturally occurring Factor C variants can be constructed using base substitutions, additions, or deletions to produce proteins having Factor C activity. Non-naturally occurring Factor C variants can differ from naturally occurring Factor C by as much as 50, 75, 80, 85, 90, 95, 97, 98, or 99%, as determined using the Blast2 alignment program (Blosum62, Expect 10, standard genetic codes). Fragments of native and recombinantly produced Factor C that retain Factor C activity also can be used in the practice of the invention. Thus, “Factor C” as used herein includes naturally and non-naturally occurring proteins and protein fragments that have the properties described above.
Methods of producing proteins recombinantly are well known in the art and generally involve culturing a host cell comprising an expression vector encoding the Factor C protein in a supernatant under conditions such that the protein is expressed. The expressed protein can be recovered or, preferably, the supernatant comprising the expressed protein is used directly as the source of recombinant Factor C. Thus, “purified Factor C” specifically includes a supernatant which comprises recombinant Factor C. Host cells useful for the production of recombinant Factor C include, without limitation, yeast cells and insect cells. Recombinant production of Carcinoscorpius rotundicauda Factor C in Pichia pastoris and Saccharomyces cerevisiae host cells is specifically disclosed in U.S. Pat. No. 5,985,590.
A particularly preferred method of obtaining recombinant Factor C is to produce the protein in a baculovirus system, as described in Examples 2-5. Briefly, a Factor C coding sequence is cloned into pFASTBAC1. The resultant recombinant plasmid is transformed into DH10BAC competent cells that contain a bacmid with a mini-attTn7 target site and a helper plasmid. In the presence of transposition proteins provided by the helper plasmid, the mini-Tn7 transposable element on the pFASTBAC plasmid can transpose to the mini-attTn7 target site on the bacmid. Colonies containing recombinant bacmids are identified by disruption of the lacZa gene. High molecular weight DNA is prepared from selected DH10BAC clones containing the recombinant bacmid. This DNA is then used to transfect Sf9 insect cells, which will then secrete recombinant Factor C. A particularly surprising discovery of the present invention is that culture medium containing the secreted Factor C, which has been separated from the cultured cells, can be used together with a surfactant (described below) as a reagent in endotoxin assays without further purification.
Surfactant
Reagents of the invention also comprise a surfactant. Surfactants useful in the practice of the invention include those described in U.S. Pat. No. 4,322,217, although other surfactants also can be used. Useful surfactants include amphoteric surfactants that contain both an anionic and cationic group in their structure. Illustrative are the sulfobetaines represented by Formula A: 




    
    
        R1 is an alkylene radical having from 1 to about 4 carbon atoms,
        Y is any non-deleterious, chemically suitable substituent including (1) hydrogen, (2) substituted or unsubstituted lower alkyl, e.g., containing 1 to 4 carbon atoms such as methyl, ethyl, propyl, or hydroxy etc.;
        R2 and R3 are each selected from substituted or unsubstituted lower alkyl containing 1 to 4 carbon atoms, e.g., such as methyl, ethyl, propyl, hydroxy ethyl, hydroxy methyl, hydroxy propyl, etc.
        n=0 or 1,
        when n=0, R4 is substituted or unsubstituted alkyl, e.g., containing about 8 to about 18 carbon atoms, and
        when n=1, R4 is an alkylene radical having from about 1 to about 6 carbon atoms, R5 is a substituted or unsubstituted alkyl, e.g., containing about 8 to about 18 carbon atoms.
    
    


The term “alkylene” encompasses both polymethylene radicals and other divalent saturated aliphatic radicals. Thus, there may be branching in the linkage provided by the alkylene radical. The term “lower” means a radical containing 1 to 4 carbon atoms.
Sulfobetaines that can be used in the reagent of the present invention are known in the art and have been described as zwitterionic surfactants. The preparation of such compounds is described, for example, in Fernley, J. Am. Oil Chem. Soc. 55, 98-103 (1978) and U.S. Pat. No. 3,280,179. In preferred sulfobetaine surfactants, R2 and R3 in the above structure are methyl. It is also preferred that R1 be propylene.
One type of useful sulfobetaine surfactant has the above structure wherein n equals 0 and R4 is an alkyl radical having from about 8 to 18 carbon atoms, preferably a straight chain alkyl radical. For these sulfobetaine surfactants, a convenient source of the R4 component is tallow fatty alcohol, which consists of a mixture of various chain lengths, with a typical composition being approximately 66 percent C18, 30 percent C16, and 4 percent C14 and others. Another convenient source is the middle cut of distilled coconut fatty alcohol, which also consists of a mixture of various chain lengths, with a typical composition being approximately 66 percent C12, 23 percent C14, 9 percent C16 and 2 percent C10.
Specific sulfobetaine surfactants of the above structure wherein n equals 0 are set forth in U.S. Pat. No. 3,539,521. A particularly preferred surfactant of this type is N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, which is commercially available from Calbiochem-Behring Corporation under the trademark ZWITTERGENT 3-14.
Another type of useful sulfobetaine surfactant has the above structure wherein n equals 1 and R4 is an alkylene radical having from about 1 to about 6 carbon atoms. In these sulfobetaines wherein n equals 1, R5 is an alkyl radical having from about 8 to about 18 carbon atoms. It is preferred that R5 be straight chain. As previously discussed, convenient sources of alkyl radicals having from about 10 to about 18 carbon atoms are tallow fatty alcohol and coconut fatty alcohol. Specific sulfobetaine surfactants of the above structure wherein n equals 1 are set forth in U.S. Pat. No. 3,280,179.
Particularly preferred sulfobetaine surfactants are 3-(N,N-dimethyl-N-acylamidopropylammonio)-2-hydroxy-propane-1-sulfonates, wherein the acyl group is derived from tallow fatty alcohol or coconut fatty alcohol, with coconut fatty alcohol preferred. It will be recognized by those skilled in the art that, in the normal preparation of these derivatives of tallow or coconut fatty alcohols, a mixture of sulfobetaines with varying carbon chain lengths for the acyl groups will result. As previously discussed, these fatty alcohols contain, for the most part, carbon chain lengths that will provide acyl groups with the desired number of carbon atoms, i.e., from about 8 to about 18 carbon atoms. Thus, these mixtures obtained from tallow or coconut fatty alcohols are useful in providing the sulfobetaine surfactant for reagents of the present invention.
A material of this type particularly preferred for use in reagents of the present invention is N-cocoamido-propyl-N,N-dimethyl-N-2-hydroxypropyl sulfobetaine. An example of this is LONZAINE CS, which is commercially available from Lonza, Inc., Fair Lawn, N.J.
Other amphoteric surfactants include the N-long chain alkyl aminocarboxylic acids illustrated by Formula B: 




N-long chain alkyl iminodicarboxylic acids illustrated by Formula C: 




and N-long chain alkyl or amido betaines illustrated by Formula D: 




where R1, R2, R3, R4, Y, and n have the same meaning as they have in Formula A, M is hydrogen or a salt-forming metal, and Y′ has the same meaning as Y in Formula A. Y and Y′ may be the same or different. Examples of specific amphoteric detergents are N-alkylbeta-aminopropionic acid, N-alkyl-beta-iminodipropionic acid, and N-alkyl-N,N-dimethyl glycine; the alkyl group may be, for example, that derived from coco fatty alcohol, lauryl alcohol, myristyl alcohol (or a lauryl-myristyl mixture), hydrogenated tallow alcohol, cetyl, stearyl, or blends of such alcohols. The substituted aminopropionic and iminodipropionic acids are often supplied in the sodium or other salt forms, which also can be used in reagents of the invention.

Specific examples include cocobetaine sold by Witco Chemical Corporation under the name EMCOL CC 37-18, cocoamidopropyl betaine sold by Lonza Inc. under the name LONZAINE CO, and disodium N-tallow-beta-iminodipropionate sold by Henkel Corporation under the name of DERIPHAT 160.
Examples of other amphoteric detergents are the fatty imidazolines such as those made by reacting a long chain fatty acid (e.g., of 10 to 20 carbon atoms) with diethylene triamine and monohalocarboxylic acids having 2 to 6 carbon atoms, e.g. 1-coco-5-hydroxyethyl-5-carboxymethylimidazoline. Specific examples include cocoimidazoline, which is commercially available under the name AMPHOTERGE K-2 from Lonza, Inc., and capric dicarboxy imidazoline, which is commercially available under the name AMPHOTERGE KJ-2 from Lonza, Inc.
Other examples of useful surfactants include anionic synthetic surfactants, generally described as those compounds that contain hydrophilic and lipophilic groups in their molecular structure and ionize in an aqueous medium to give anions containing both the lipophilic group and hydrophilic group. The alkyl aryl sulfonates, the alkane sulfates, and sulfated oxyethylated alkyl phenols are illustrative of the anionic type of surface active compounds.
The alkyl aryl sulfonates are a class of synthetic anionic surface active agents represented by Formula E:

(R6)n1-(Y)Ar(SO3M)n2  (E)

In Formula E, R6 is a straight or branched chain hydrocarbon radical having from about 1 to about 24 carbon atoms, at least one R6 having at least 8 carbon atoms; n1 is from 1 to 3; n2 is from 1 to 2; Ar is a phenyl or a naphthyl radical, and Y and M have the same meaning as in Formula B. R6 can be, for example, methyl, ethyl, hexyl, octyl, tetraoctyl, iso-octyl, nonyl, decyl, dodecyl, octadecyl, and the like.
Compounds illustrative of the alkyl aryl sulfonates include sodium dodecylbenzene sulfonate, sodium decylbenzene sulfonate, ammonium methyl dodecylbenzene sulfonate, ammonium dodecylbenzene sulfonate, sodium octadecylbenzene sulfonate, sodium nonylbenzene sulfonate, sodium dodecylnaphthalene sulfonate, sodium hetadecylbenzene sulfonate, potassium eicososyl naphthalene sulfonate, ethylamine undecylnaphthalene sulfonate and sodium docosylnaphthalene sulfonate.
The alkyl sulfates are a class of synthetic anionic surface active agents represented by Formula F:

R5OSO3M  (F)

where R5 and M have the same meaning as in Formula B. Compounds illustrative of alkyl sulfate class of anionic surfactants include sodium octadecyl sulfate, sodium hexadecyl sulfate, sodium dodecyl sulfate, sodium nonyl sulfate, ammonium decyl sulfate, potassium tetradecyl sulfate, diethanolamino octyl sulfate, triethanolamine octadecyl sulfate, and amrnmonium nonyl sulfate.

The sulfated oxyethylated alkylphenols are a class of synthetic anionic surface active agents represented by Formula G: 




where A is either oxygen, sulfur, a carbonamide group, a thiocarbonamide group, a carboxylic group, or a thiocarboxylic ester group, z is an integer from 3 to 8, and R5 and M have the same meaning as in Formula B. Compounds illustrative of the sulfated oxyethylated alkyl phenol class of anionic surfactants include ammonium nonylphenoxyl tetraethylenoxy sulfate, sodium dodecylphenoxy triethyleneoxy sulfate, ethanolamine decylphenoxy tetraethyleneoxy sulfate, and potassium octylphenoxy triethyleneoxy sulfate.

Other examples of useful surfactants include nonionic surface active compounds, which can be broadly described as compounds that do not ionize but acquire hydrophilic characteristics from an oxygenated side chain, such as polyoxyethylene; the lipophilic part of the molecule may come from fatty acids, phenol, alcohols, amides, or amines. The compounds are usually made by reacting an alkylene oxide, such as ethylene oxide, butylene oxide, propylene oxide and the like, with fatty acids, straight or branched chain alcohols containing one or more hydroxyl groups, phenols, thiophenols, amides, or amines to form polyoxyalkylene glycoethers and esters, polyoxyalkylene alkylphenols, polyoxyalkylene thiophenols, polyoxyalkylene amides and the like. It is generally preferred to react from about 3 to about 30, more preferably 10 to 30, moles of alkylene oxide per mole of the fatty acids, alcohols, phenols, thiophenols, amides, or amines.
Illustrative of these nonionic surfactants are the products obtained from the reaction of alkylene oxide with an aliphatic alcohol having from 8 to 18 carbon atoms, such as octyl, nonyl, decyl, octadecyl, dodecyl, tetradecyl and the like, with monoesters of hexahydric alcohols, the ester group containing 10 to 20 carbon atoms such as sorbitan monolaureate, sorbitan monooleate and sorbitan monopalmitate, with an alkyl phenol in which the alkyl group contains between 4 and 20 carbon atoms, such as butyl, dibutyl, amyl, octyl, dodecyl, tetradecyl, and the like, or with an alkyl amine in which the alkyl group contains between 1 to 8 carbon atoms.
Compounds illustrative of synthetic nonionic surfactants include the products obtained from condensing ethylene oxide or propylene oxide with the following: propylene glycol, ethylene diamine, diethylene glycol, dodecyl phenol, nonyl phenol, tetradecyl alcohol, N-octadecyl diethanolamide, N-dodecyl monoethanolamide, polyoxyethylene (20) sorbitan monooleate sold under the name TWEEN 80 and polyoxyethylene (20) sorbitan monolaurate sold under the name TWEEN 20.
Other nonionic surfactants include long chain tertiary amine oxides corresponding to Formula H:

R5R7R8N→O  (H)

wherein R5 has the same meaning as in Formula A, and R7 and R8 are each methyl or ethyl radicals. The arrow in the formula is a conventional representation of a semi-polar bond. Examples of amine oxides suitable for use in this invention include dimethyldodecylamine oxide, dimethyloctylamine oxide, dimethyldecylamine oxide, dimethyltridecylamine oxide, and dimethylhexadecylamine oxide.

Cationic surface active agents can also be used as surfactants. Such agents are those surface-active compounds which contain an organic hydrophobic group and a cationic solubilizing group. Typical cationic solubilizing groups are amine and quaternary groups. Such cationic surface-active agents are represented by Formula I: 




wherein R5, Y, and Y′ have the same meaning as in Formula C.

Other examples of suitable synthetic cationic surfactants include the diamines such as those of Formula J:

R9NHC2H4NH2  (J)

wherein R is an alkyl group of about 12 to 22 carbon atoms, such as N-2-aminoethyl stearyl amine and N-2-aminoethyl myristyl amine; amide-linked amines such as those of Formula K:

R5CONHC2H4NH3  (K)

such as N-2-amino ethylstearyl amide and N-amino ethyl myristyl amide; quaternary ammonium compounds wherein typically one of the groups linked to the nitrogen atom are alkyl groups which contain 1 to 3 carbon atoms, including such 1 to 3 carbon alkyl groups bearing inert substituents, such as phenyl groups and there is present an anion such as halogen, acetate, methylsulfate, etc. Typical quaternary ammonium compounds are ethyl-dimethylstearyl ammonium chloride, benzyl-dimethyl-stearyl ammonium chloride, benzyldimethyl-stearyl ammonium chloride, trimethyl stearyl ammonium chloride, trimethylcetyl ammonium bromide, dimethylethyl dilaurylammonium chloride, dimethyl-propyl-myristyl ammonium chloride, and the corresponding methosulfates and acetates.

Another suitable cationic surfactant is represented by Formula L: 




wherein R5 has the same meaning as in Formula A and each a is an integer from 1 to 15. An example is the polyethylene glycol amine of hydrogenated tallow wherein R5 represents the tallow radical and a+a has an average value of 5.

Other useful surfactants for use in the Factor C-surfactant reagent include Triton X-100, Triton X-114, octyl-beta-D-thioglucoside (OTG, Amresco #J575), Genapol C-100 (an alkyl polyoxyethylene C12E1O; Calbiochem #345794), Tween 20, and Tween 80.
Factor C-Surfactant Reagent
To form a reagent of the invention, purified Factor C and a surfactant are combined in an aqueous solution. A suitable buffer solution for this purpose contains 30 mM Tris, pH 8.0, 30 mM NaCl, and 0.3% lactose. The concentration of purified Factor C in a reagent of the invention preferably ranges from 0.03-3 μg/ml. The concentration of surfactant in the reagent of the invention preferably ranges from 0.001-0.003%. Optimum concentrations of purified Factor C and of the surfactant will vary, depending, for example, on the purity of the Factor C and the particular surfactant. Optimum concentrations of purified Factor C and of surfactant can be determined using routine testing. In the assay exemplified in Examples 1 and 13, the optimal concentration of the surfactant ZWITTERGENT 3-14 is 0.0025%.
Endotoxin Assay
The reagent described above can be used in an assay to detect endotoxin in a test sample. The test sample can be any sample in which it would be useful to detect endotoxin, including water, aqueous solutions such as buffers, pharmaceutical preparations (e.g., vaccines, intravenous fluids, drug preparations), biomedical imaging reagents (e.g., dyes, radioactive solutions), enzyme preparations (e.g., collagenase), tissue culture media, beverages, blood, urine, cerebrospinal fluid, lymph, serum, and solutions formed by incubating water or an aqueous solution with a solid sample, such as a foodstuff or a surgical glove.
Either a two-step or a one-step assay can be performed. To perform the two-step assay (Example 1), a test sample is incubated with a reagent containing purified Factor C and a surfactant, then a Factor C substrate that will generate a detectable signal upon cleavage is added. To perform the one-step assay (Example 13), endotoxin, the reagent containing purified Factor C and a surfactant, and a substrate are added together.
Substrates that will generate a fluorescent signal upon cleavage are particularly preferred, such as N-t-BOC-Asp(Obzl)-Pro-Arg-7-Amido-4-methyl coumarin (“DPR,” Bachem 1-1560.0050) and N-t-BOC-Val-Pro-Arg-7-Amido-4-methyl coumarin (“VPR,” Bachem 1-1120.0050). Concentrations of DPR generally range from about 10-100 mM, 25-100 mM, 5-100 mM, 75-100 mM, 25-75 mM, 50-75 mM, 25-50 mM, or 50-75 mM. Concentration of VPR generally range from about 50-200 mM, 50-150 mM, 50-100 mM, 50-75 mM, 75-150 mM, or 75-100 mM.
The optimum concentration for a particular substrate varies. For example, the optimum concentration for DPR is 50 mM, whereas the optimum concentration for VPR is 100 mM. Moreover, the particular substrate can be chosen depending on the endotoxin levels expected to be present in the test sample. The VPR substrate has a better linear range at higher endotoxin concentrations, and the DPR substrate has a better linear range at lower endotoxin concentrations. Thus, DPR can be used in assays in which higher sensitivity is desired, whereas VPR is the preferred substrate when higher endotoxin levels are expected to be present and a lower sensitivity is required.
Fluorescence can be measured using any means known in the art. If a fluorimeter is used with the DPR or VPR substrates described above, the excitation is set at 360, 380, 390, or 395 nm (slit width of 5-40 nm) and the emission is measured at 440 or 460 nm (slit width of 2.5-40 nm). Measurement can be either qualitative or quantitative, by reference to a standard endotoxin concentration curve.
The assay can be carried out in a clear vessel, such as a glass or polystyrene tissue culture plate, or can be carried out in a black vessel (e.g., a black 96-well microplate). If desired, the assay can be adapted for high-throughput screening of multiple samples using, for example, 96-well microtiter plates.
Kits
The invention also provides a kit for use in detecting endotoxin. The kit comprises a purified horseshoe crab Factor C protein and a surfactant, as described above. Instructions for using the reagent to detect endotoxin can be included. Kits also can include a Factor C substrate for use in the assay.
All patents, patent applications, and references cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided for purposes of illustration only and are not intended to limit the scope of the invention.
EXAMPLE 1
Two-Step Endotoxin Assay
Endotoxin was pre-incubated with culture medium containing recombinant Carcinoscorpius rotundicauda Factor C obtained from recombinant Factor C-producing Sf9 cells for 1 hour in assay buffer (30 mM Tris, pH 8.0, 30 mM NaCl, 0.3% lactose, and 0.0025% Zwittergent 3-14). The substrate N-t-Boc-Asp(Obzl)-Pro-Arg-7-Amido-4-methyl coumarin (DPR-coumarin) was added to the mixture to a final concentration of 50 mM. The fluorescence generated from the cleavage of the substrate by activated Factor C was measured after 15-20 minutes. The results are shown in FIG. 4. Results of a similar assay carried out using the substrate N-t-Boc-Val-Pro-Arg-7-Amido-4-methyl coumarin at a final concentration of 100 mM are shown in FIG. 5.
EXAMPLE 2
Transfection of Bacmid DNA-Factor C into Sf9 Cells and Harvesting of Recombinant Viral Supernatant
Sf9 cells are seeded at a density of 5×106 per 35 mm well in a 6-well tissue culture plate in insect cell culture medium (ICCM) containing 50 U/ml penicillin and 50 mg/ml streptomycin. Insect-Xpress (BioWhittaker Cat. #04-10270) or Sf-900 SFM are suitable media; however, any comparable medium in which SF9 cells grow can be used. The plates are incubated at 27° C. for 1 hour to allow the cells to settle. Meanwhile, the following are prepared (each per well): (1) 7 mg of bacmid-Factor C DNA in 100 ml ICCM (without antibiotics) and (2) 6 ml CELLFECTIN (Gibco-BRL) plus 100 ml ICCM (without antibiotics). Solutions (1) and (2) are mixed gently and incubated at room temperature for one hour. To this mixture, 0.8 ml of ICCM (without antibiotics) is added.
The adhered Sf9 cells are washed gently with 2 ml ICCM (without antibiotics). One ml of CELLFECTIN-DNA complex is added to each well and incubated at 27° C. for 5 hours. The transfection mixtures are removed completely, and 2 ml of ICCM containing antibiotics is added. After incubation at 27° C. for 96 hours, the cell culture supernatant is harvested. The supernatant is clarified by centrifugation at 5000 rpm for 10 minutes in a SIGMA 3K10 swing-out rotor, Nr. 11133. The supernatant, containing the recombinant baculovirus, is stored at 4° C.
EXAMPLE 3
Amplification of Recombinant Viral Stock
Virus amplification can be done with Sf9 cells either in a monolayer or in suspension. For monolayer culture, the medium is decanted from a culture of 80% confluent one day-old Sf9 cells in a 75 cm2 tissue culture flask. The cell monolayer is infected with 1 ml of virus (Example 2) using an MOI of 0.1-1. The virus stock is sterile-filtered using a Millipore GV millex filter (yellow; low-protein binding). Calculation of the viral inoculum is as follows: 



  
    volume
    ⁢
    
       
    
    ⁢
    of
    ⁢
    
       
    
    ⁢
    inoculum
  
  =
  
    
      MOI
      ×
      total
      ⁢
      
         
      
      ⁢
      cell
      ⁢
      
         
      
      ⁢
      number
    
    
      virus
      ⁢
      
         
      
      ⁢
      titer
      ⁢
      
         
      
      ⁢
      in
      ⁢
      
         
      
      ⁢
      pfu
      ⁢
      
        /
      
      ⁢
      ml
    
  




Thus, for 1×107 cells, at a virus titer of 2×107 pfu/ml, the volume of the inoculum is 0.5 ml. The volume of the inoculum is adjusted to 1 ml with ICCM before it is introduced to the cells.
The flask is rocked several times to ensure that the cell monolayer is completely covered by the 1 ml viral inoculum. The flask is then incubated at 27° C. for one hour without rocking. After the incubation, the flask is placed upright, and 14 ml of fresh ICCM is added. The flask is then incubated for 3 days at 27° C.
The culture supernatant is harvested into sterile pyrogen-free tubes and centrifuged at 2000 rpm for 10 minutes at 4° C. using a Sigma 3K10 swing out rotor. For immediate use, the viral stock is stored at 4° C. For long-term storage, the viral stock is placed at −80° C.
For suspension culture, use log-phase grown Sf9 cells with viability >95% for vial amplification. Cell density and viability are determined. Sf9 cells are diluted with fresh InsectXPRESS to 1×106 cells per ml. Virus to be amplified is added to the Sf9 culture, at an MOI of 0.02 (virus/cell), as follows: 



  
    
      Vol
      .
      
         
      
      ⁢
      of
    
    ⁢
    
       
    
    ⁢
    inoculum
    ⁢
    
       
    
    ⁢
    virus
    ⁢
    
       
    
    ⁢
    stock
    ⁢
    
       
    
    ⁢
    
      (
      ml
      )
    
  
  =
  
    
      
        (
        
          Total
          ⁢
          
             
          
          ⁢
          cells
          ⁢
          
            /
          
          ⁢
          ml
        
        )
      
      ×
      
        (
        
          Total
          ⁢
          
             
          
          ⁢
          
            Vol
            .
            
               
            
            ⁢
            of
          
          ⁢
          
             
          
          ⁢
          culture
        
        )
      
      ×
      
        (
        MOI
        )
      
    
    
      (
      
        Virus
        ⁢
        
           
        
        ⁢
        titer
        ⁢
        
           
        
        ⁢
        in
        ⁢
        
           
        
        ⁢
        pfu
        ⁢
        
          /
        
        ⁢
        ml
      
      )
    
  




Spinner flasks are incubated for three days at 27° C.+/−2° C., with constant stirring at 90-120 rpm. The cell culture is transferred to sterile centrifuge bottles and centrifuged at 2000 rpm for 10 minutes at 4° C. The supernatant is collected into a sterile container and discard the cell pellet. The supernatant is filtered through a 0.45 μm filter into a sterile container. The supernatant is further filtered through a sterile, 0.2 μm filter into a sterile container. The finished virus stock is stored protected from light at 2-8° C. A titer determination is performed as explained in Example 4.
EXAMPLE 4
Titration of Baculovirus 
Virus titer (Plaque Forming Units (PFU) per ml) can be determined with plaque assay, end-point dilution, or other viral titer kits (e.g., BacPAK Baculovirus Rapid Titer Kit by Clontech #K1599-1). Plaque assay is performed in immobilized monolayer culture. Plaque assays to be are performed are determined as follows:

Plaque assay number=Number of viral dilution×4 (quadruplicates)+2 positive and 2 negative controls.

For example, 105, 106, 107, 108 for viral dilution, each dilution in quadruplicate wells, would be a 16-well plaque assay. Include two positive controls and two negative controls in the plaque assay, for a total of 20 wells.

Prepare a cell suspension of 5×105 cells per ml density in 10% FBS serum media. Seed 6-well plates at 2 ml per well with above cell suspension. Cells are allowed to attach two hours before experiments. Cells are  ̃80% confluent.
Prepare 10-fold serial viral dilutions using 4.5 ml media and 0.5 ml of viral stock in sterile 15-ml tubes. Aspirate the medium from each well. Add viral dilution 1 ml per well and incubate at 27° C. for 2 hours.
Warm 1.3× SF-900II (Gibco-BRL #10967-032) to 27° C. Melt 4% agarose (Gibco 18300-012) and keep in 50° C. water-bath. Add 1.3× media and 4% agarose in 3:1 ratio to make 1% agarose in culture media. Keep this agar in a 40° C. water-bath (for example, in a beaker that can be brought to a hood).
Working quickly so that the agarose mixture does not solidify, aspirate the viral solution from the cell monolayers. Add 2 ml 1% agarose to each well. Let the plates sit in the hood with the covers slightly open for 10 minutes. Invert the plates and put them in a sealed box with damp paper towels to provide humidity. Incubate at 27° C. for 5-7 days, or until plaques are well formed.
At day 7 of post-infection, add 0.5 ml 0.033% neutral red (in water), incubate 3 minutes, and discard. Alternatively, add 0.5 ml 0.1% trypan blue (in water), rock to cover, sit 3 minutes, and discard. Incubate the plates for 2 hours. With the help of a light box and a magnifier, count the plaques on each plate. Virus titer is calculated using the following formula:

Virus titer (pfu/ml)=# of plaques×viral dilution.

EXAMPLE 5
Infection of Sf9 Cells for Recombinant Factor C Production in Serum-Free Cultures
High viability Sf9 cells are used (e.g., viability of 95-100% in serum-free conditions). Culture log-phase Sf9 cells to a cell density of between 1.5×106 and 2.5×106 cells per ml in suspension culture. Recombinant factor C production also can be carried out by infecting Sf9 cells grown in monolayer culture. Determine cell density and viability. Dilute Sf9 cells with fresh InsectXPRESS to 1.5×106 cells per ml. Add recombinant baculovirus high titer stock to the Sf9 culture, at an MOI of 1 (virus/cell), as follows: 



  
    
      Vol
      .
      
         
      
      ⁢
      of
    
    ⁢
    
       
    
    ⁢
    inoculum
    ⁢
    
       
    
    ⁢
    HTS
    ⁢
    
       
    
    ⁢
    
      (
      ml
      )
    
  
  =
  
    
      
        (
        
          Total
          ⁢
          
             
          
          ⁢
          cells
          ⁢
          
            /
          
          ⁢
          ml
        
        )
      
      ×
      
        (
        
          Total
          ⁢
          
             
          
          ⁢
          
            Vol
            .
            
               
            
            ⁢
            of
          
          ⁢
          
             
          
          ⁢
          culture
        
        )
      
      ×
      
        (
        MOI
        )
      
    
    
      (
      
        Virus
        ⁢
        
           
        
        ⁢
        Titer
        ⁢
        
           
        
        ⁢
        in
        ⁢
        
           
        
        ⁢
        pfu
        ⁢
        
          /
        
        ⁢
        ml
      
      )
    
  




Harvest culture supernatant seventy-two hours post-infection. Transfer the cell culture to sterile centrifuge bottles and centrifuge at 2000 rpm for 10 minutes at 4° C. Collect the supernatant into a sterile container and discard the cell pellet. Filter the supernatant through a 0.45 μm filter into a sterile container. Further filter supernatant through a sterile, 0.2 μm filter into a sterile container. This culture supernatant, which contains recombinant Factor C, can be stored at 4° C. for up to one year and can be used directly to form a reagent for endotoxin detection in accordance with the present invention.

EXAMPLE 6
Culture and Subculture of Sf9 Cells Adapted in Serum-Free Medium
Sf9 cells grow at 27° C. without CO2. Maximal aeration is preferred. If spinner culture bottles are used, loosen the side screw-caps to increase aeration. Sf9 cells adapted to grow in serum-free medium should be subcultured at 4-5 days intervals on a routine basis.
Subculture at 5.0×105 cells/ml in a 250 ml spinner or shake flask containing 100 ml of medium with loosened caps to provide maximal aeration. Stir the culture at 90-100 rpm. Cell viability should be above 90%.
Remove 4-5 day old cultures from the 27° C. incubator and swab with 70% alcohol. Take an aliquot of cell suspension to determine cell viability and total cell count using trypan blue dye. Determine the cell dilution necessary to obtain 5×105 cells/ml. Swirl the stock culture flask several times and remove the appropriate volume of cell suspension based on the calculation. Inoculate a new 250 ml spinner flask containing enough pre-warmed fresh medium necessary to make up the final culture volume to 100 ml. Place the cultures back into the incubator. Set the stirring speed to 90-100 rpm.
EXAMPLE 7
Cryopreservation of Serum-Free Cultures of Sf9 Cells
Grow Sf9 cells either in a monolayer or in suspension. Harvest the cells at mid-log phase of growth (about 2 days) at a viability of >90%. Keep conditioned medium on ice and sterile-filter.
Determine the cell viability using the trypan blue dye exclusion method. Calculate the required volume of cryopreservation medium (7.5% DMSO in 50% fresh serum-free ICCM medium and 50% conditioned medium; sterile-filtered) to yield a final density of 1-2×107 cells/ml. Hold the cryopreservation mixture on ice. Meanwhile, centrifuge cells from the suspension for 3 minutes at 800 rpm/500× g. Discard the sample if a lot of cells are unpelleted and in the supernatant.
Resuspend the cell pellet in the determined volume of chilled cryopreservation mix to obtain 1-2×107 cells/ml. Quickly dispense 1-1.5 ml aliquots of cell suspension into cryovials. Freeze the cells at −70 to −90° C. for 4 hours on the upper gaseous phase of liquid nitrogen. Plunge the vials into liquid nitrogen.
EXAMPLE 8
Recovery of Cryopreserved Serum-Free Sf9 Cells
Thaw vial by rapid agitation in 37° C. water bath just until melted. Remove the vial from the water bath and swab with sterile 70% isopropanol. Open vial, remove contents of the vial into a sterile centrifuge tube of appropriate size; and dilute the cell suspension with 5 volumes of InsectXPRESS medium (or equivalent) that has been equilibrated to 27° C.+/−2° C. in an incubator. Centrifuge the diluted suspension at 125× G for 10 minutes. Discard the fluid and resuspend the cells in a volume of culture medium equal to fluid discarded. Determine cell count and viability. Adjust viable cell density to between 2×105 cells per ml and 3×105 cells per ml using serum free medium. Transfer flask in a 27° C.+/−2° C. incubator on a rotary platform shaker set for continuous shaking and 100-125 revolutions per minute. When the viable cell count of the culture doubles at least every 26 hours, the culture may be expanded as described in Example 6.
EXAMPLE 9
Adaptation of Sf9 Cells to Suspension Culture
Six to ten confluent 75 cm2 monolayer T-flasks of Sf9 cells are required to initiate a 100 ml suspension culture. Dislodge cells from the bottom of the flasks by holding a flask in one hand and tapping it against the palm of the other hand. Pool the cell suspension and perform a viability count.
Dilute the cell suspension to approximately 5×105 viable cells/ml at room temperature using complete serum-supplemented or serum-free growth medium contained in a 250 ml spinner flask. The top of the stirrer blade may be slightly above the cell suspension. This provides additional aeration. The side arm caps should be loosened by one-quarter turn.
Incubate the cells at 27° C. at a constant stirring rate of 100 rpm. Subculture when the viable cell count reaches 1-2×106 cells/ml (3-7 days post-seeding), increase stirring speed by 5 rpm per passage until culture viability is >80%. Repeat until a stirring speed of 100 rpm is achieved for the spinner flask or 130-140 rpm is achieved for the shaker flask cultures. At this point, reduce seeding density to 3×105 cells/ml during subculture.
If large clumps of cells persist (e.g., >10 cells per clump), let the spinner/shaker flask stand 2-3 minutes without stirring prior to subculturing. This allows larger clumps to settle to the bottom. Pool the upper one-third of the suspension cells for counting and seeding into new cultures. This procedure selects for a cell population that grows as single cells. It may be necessary to repeat this step 2-3 times until clumping is reduced.
EXAMPLE 10
Effect of Different Detergents and Different Concentrations of Detergents on the Recombinant Factor C Assay
Materials and Methods
The following reagents were purchased from the manufacturers indicated: Zwittergent 3-14 (Calbiochem, Cat# 693017), Tween 20 (ICN, Cat# 194841), Tween 80 (ICN, Cat# 194842), Triton X-114 (ICN, Cat# 193971), ), n-Octyl-beta-D-thioglycopyranoside (OTG, Amresco, Cat# J575), and Triton X-100 (ICN, Cat# 194854). The recombinant Factor C (rFC) assay was carried out using 20 μl of each detergent ( ̃10× of final concentration), 150 μl of 1 EU/ml EC-6, 10 μl of rFC supernatant 031901I, 20 μl of 300 mM Tris, pH 8.0, and 20 μl of 0.55 mM DPR-coumarin substrate. The results of the assay were read at 38° C. using a Cytofluor reader set at 390/440 nm, 5 minutes per cycle, for 1 hour. The data were graphed after 30 minutes.
Results
As shown in FIGS. 1-3 and 9-11, low concentrations of Zwittergent 3-14, Tween 20, Tween 80, Triton X-114, OTG, Genapol C-100 and Triton X-100 enhanced the rFC assay 2-7 fold. The enhancing concentration range for Zwittergent 3-14, Triton X-114, OTG and Triton X-100 is narrow, while the enhancing concentration range for Tween 20, Tween 80 and Genapol C-100 is wide.
Table 1 summarizes the results obtained by testing nine detergents. These detergents can be divided into three groups: detergents that have a narrow concentration range for enhancing recombinant Factor C activity, detergents that have wide concentration range for enhancing recombinant Factor C activity, and detergents that inhibit recombinant Factor C activity.











TABLE 1









Detergent concentration range for
CMC













Detergents
rFC enhancing
rFC inhibition
(mM)
MW
Category















Group 1







Zwittergent
0.001-<0.004%
 >=0.004%
0.1-0.4
363.6
ZW


Triton X-100
0.001-<0.008%
 >=0.008%
0.2-0.9
631
Non-ionic


Triton X-114
0.001-<0.016%
 >=0.016%
0.35
537
Non-ionic


OTG
0.031-<0.5% 
  >=0.5%
9
308.4
Non-ionic


Group 2


Genapol C-100
0.001-0.5%  
   >0.5%
/
627
Non-ionic


Tween 20
0.001-0.5%  
  >0.5%
0.059
1228
Non-ionic


Tween 80
0.001-0.25%  
  >0.25%
0.012
1310
Non-ionic


Group 3


Deoxycholic Acid
/
>=0.0004%
1.5
414.6
Ionic


SDS
/
>=0.0004%
2.3
288.5
Ionic









The enhancing/inhibiting effect of Zwittergent 3-14 was also tested using purified Limulus factor C (FIG. 6). A similar enhancing/inhibiting effect was observed, although the relative factor C activity ratio, the enhancing and inhibiting detergent concentrations were different from those in the recombinant Factor C assay.
EXAMPLE 11
Antisera Against Limulus Factor C
We isolated the factor C from Limulus and generated antisera against the protein. The Limulus lysate was first separated with a S-100 gel filtration followed by cation-ion exchange column. The fractions were assayed for factor C activity using the N-t-BOC-Val-Pro-Arg 7-Amido-4-methyl coumarin substrate. The Limulus factor C was purified 73 fold with 80% homogeneity as determined by enzyme assay and SDS-PAGE. The molecular weight of Limulus factor C was 117 kDa by SDS-PAGE under non-reducing conditions. Reduced SDS-PAGE showed two major bands of 79 kDa and 40 kDa, corresponding to the heavy and light chain. Tryptic peptide sequencing revealed that the Limulus factor C sequence closely matched the Asian species, with >90% sequence identity. A specific polyclonal antibody against the Limulus factor C was generated in rabbits. The anti-factor C purified IgG inhibited the activation of factor C by LPS but had no effect on the protease activity once the factor C was activated by LPS. The factor C antisera demonstrated the importance of this protein in the initial recognition of LPS by Limulus lysate.
EXAMPLE 12
Increase in Sensitivity in Endotoxin Detection in the Presence of Surfactant
A one-step, 1-hour endpoint assay was performed. Endotoxin 055:B5, 0.01, 0.1, 1, and 10 EU/ml were used for the standard curve. Blank and endotoxin standards (100 μl each) were added to a 96-well plate. Recombinant Factor C supematant, assay buffer (150 mM NaCl, 150 mM Tris, pH 8.0, and 1.5% lactose, with or without 0.0125% Zwittergent) and substrate solution (0.2 mM in water) were mixed at 1:4:5 ratios. This mixture was added into each blank and endotoxin standard. The fluorescence was recorded at time zero and time 1 hour. The difference in fluorescences (delta fluorescence) were normalized with the blank. Normalized delta fluorescence was then graphed against endotoxin concentration in a log-log scale. Each data point is the result of duplicate assays.
The results are shown in FIG. 7. The endotoxin detection sensitivity increased 10-fold when surfactant was included.
EXAMPLE 13
One-Step Endotoxin Assay
A one-step endotoxin assay can be carried out in a 96-well plate, using 100 μl each of blank and endotoxin standards. One hundred microliters of a mixture of recombinant Factor C supernatant (Baculovirus-infected Sf9 cell culture medium), buffer (150 mM Tri, pH 8.0, 150 mM NaCl, 1.5% beta-lactose, and 0.0125% Zwittergent 3-14) and fluorogenic substrate (DPR-coumarin, 0.2 mM) at a ratio of 1:4:5 is added to wells of the plate. The plate is incubated at 37° C. for 1 hour. Excitation and emission are read at 390 and 440 nm, respectively, in a fluorescence microplate reader.
The results of a one-step recombinant Factor C endotoxin assay are shown in FIG. 8.

## Claims

1. A reagent for increasing sensitivity of enzymatic endotoxin detection, comprising:
a purified horseshoe crab Factor C protein; and 
a surfactant, 

wherein the purified horseshoe crab Factor C protein is enzymatically active in the presence of the surfactant and wherein the surfactant is present in an amount which increases the sensitivity of an enzymatic endotoxin detection reaction relative to a reagent containing the purified horseshoe crab Factor C protein but which does not comprise the surfactant.

2. The reagent of claim 1 wherein the surfactant is selected from the group consisting of:
I amphoteric surfactants represented by the following formulae 




wherein R1 is an alkylene radical having from 1 to 4 carbon atoms; 
Y and Y′ are each hydrogen or a substituted or unsubstituted lower alkyl; 
R2 and R3 are each a substituted or unsubstituted lower alkyl; 
n is 0 or 1, wherein when n is 0, R4 is alkyl containing from about 8 to about 18 carbon atoms, 
and when n is 1, R4 is an alkcylene radical having from 1 to about 6 carbon atoms; 
R5 is a substituted or unsubstituted alkyl; and 
M is hydrogen or a salt-forming metal; 
(II) anionic surfactants represented by the following formulae: 


(R6)n1-(Y)Ar(SO3M)n2  (E) 



wherein R6 is a straight or branched chain hydrocarbon radical having from about 1 to about 24 carbon atoms, wherein at least one R6 has at least 8 carbon atoms; 
n1 is from 1 to 3; 
n2 is from 1 to 2; 
Ar is a phenyl or a naphthyl radical; and 
Y and M have the same meaning as in Formula (B); 

 R5OSO3M  (F)
wherein M has the same meaning as in Formula (B); 
(III) cationic surfactants represented by the following formula: 





wherein 
A is either oxygen, sulfur, a carbonamide group, a thiocarbonamide group, a carboxylic group, or 
a thiocarboxylic ester group; 
z is an integer from 3 to 8; and 
M has the same meaning as in Formula (B); 
(IV) nonionic surfactants represented by the following formula: 


R5R7R8N→O  (H) 



wherein 
R5 has the same meaning as in Formula (A); 
R7 and R8 are each methyl or ethyl; 
(IV) cationic surfactants represented by the following formulae: 






wherein R5, Y, and Y′ have the same meaning as in Formula C;


R9NHO2H4NH2  (J) 


wherein R is an alkyl group of about 12 to 22 carbon atoms;


R5CONHC2H4NH3  (K) 


wherein R5 has the same meaning as in Formula (A); 




wherein R5 has the same meaning as in Formula A and each a is an integer from 1 to 15; and
(V) those nonionic surfactants selected from the group consisting of the condensation product of about 10 to 30 moles of ethylene oxide with the monoester of a hexahydric alcohol containing 6 carbon atoms with the ester group containing 10 to 20 carbon atoms.

3. The reagent of claim 1 wherein the horseshoe crab is Limulus polyphemus.

4. The reagent of claim 1 wherein the horseshoe crab is Carcinoscorpius rotundicauda.

5. The reagent of claim 1 wherein the horseshoe crab is Tachypleus tridentatus.

6. The reagent of claim 1 wherein the horseshoe crab is Tachypleus gigas.

7. The reagent of claim 1 wherein the Factor C protein is made by the method of culturing a host cell comprising a vector encoding the Factor C protein in a supernatant under conditions such that the Factor C protein is expressed into the supernatant.

8. The reagent of claim 7 wherein the host cell is an Sf9 cell.

9. The reagent of claim 7 wherein the horseshoe crab is Carcinoscorpius rotundicauda.

10. The reagent of any one of claims 1 through 9 wherein the surfactant is selected from the group consisting of ZWITTERGENT 3-14, Triton X-100, Triton X-114, octyl-beta-D-thioglucoside, Genapol C-100, Tween 20, and Tween 80.

11. A method of detecting endotoxin in a test sample, comprising the steps of:
contacting a test sample with (1) the reagent of claim 1 and (2) a Factor C substrate to form a test sample-substrate-reagent mixture, wherein cleavage of the Factor C substrate generates a detectable signal; and 
assaying the test sample-substrate-reagent mixture for the presence or absence of the detectable signal, wherein an amount of the detectable signal that is increased relative to a control sample that does not contain endotoxin indicates a presence of endotoxin in the test sample.

12. The method of claim 11 wherein the horseshoe crab is Limulus polyphemus.

13. The method of claim 11 wherein the horseshoe crab is Carcinoscorpius rotundicauda.

14. The method of claim 11 wherein the horseshoe crab is Tachypleus tridentatus.

15. The method of claim 11 wherein the horseshoe crab is Tachypleus gigas.

16. The method of claim 11 wherein the Factor C protein is made by the method of:
culturing a host cell comprising a vector encoding the Factor C protein in a supernatant under conditions such that the Factor C protein is expressed into the supenatant.

17. The method of claim 16 wherein the host cell is an Sf9 cell.

18. The method of claim 15 wherein the horseshoe crab is Carcinoscorpius rotundicauda.

19. The method of any one of claims 10-18 wherein the surfactant is selected from the group consisting of ZWITTERGENT 3-14, Triton X-100, Triton X-114, octyl-beta-D-thioglucoside, Genapol C-100, Tween 20, and Tween 80.

20. The method of claim 11 wherein the Factor C substrate is selected from the group consisting of N-t-BOC-Asp(Obzl)-Pro-Arg-7-Amido-4-methyl coumarin and N-t-BOC-Val-Pro-Arg-7-Amido-4-methyl coumarin.

21. A method of detecting endotoxin in a test sample, comprising the steps of:
contacting a test sample with: 
(1) a reagent comprising (a) a surfactant in an amount which increases the sensitivity of an enzymatic endotoxin detection reaction relative to a reagent containing a purified horseshoe crab Factor C protein but which does not comprise the surfactant and (b) a recombinant Carcinoscorpius rotundicauda Factor C protein, wherein the Factor C protein is made by the method of culturing a host cell comprising a vector encoding the Factor C protein in a supernatant under conditions such that the Factor C protein is expressed into the supernatant, wherein the Factor C protein is enzymatically active in the presence of the surfactant; and 
(2) N-t-BOC-Asp(Obzl)-Pro-Arg-7-Amido-4-methyl coumarin to form a test sample-substrate-reagent mixture; and 

assaying the test sample-substrate-reagent mixture for the presence or absence of a fluorescent signal, wherein an amount of the fluorescent signal that is increased relative to a control sample that does not contain endotoxin indicates a presence of endotoxin in the test sample.

22. The method of claim 21 wherein the host cell is an Sf9 cell.

23. A kit for detecting endotoxin, comprising:
the reagent of claim 1; and 
instructions for the method of claim 11.

24. The kit of claim 23 further comprising a Factor C substrate, wherein cleavage of the Factor C substrate generates a detectable signal.

25. The kit of claim 24 wherein the Factor C substrate is selected from the group consisting of N-t-BOC-Asp(Obzl)-Pro-Arg-7-Amido-4-methyl coumarin and N-t-BOC-Val-Pro-Arg-7-Amido-4-methyl coumarin.

26. The kit of claim 23 wherein the purified Factor C is made by the method of culturing a host cell comprising a vector encoding the Factor C protein in a supernatant under conditions such that the Factor C protein is expressed into the supernatant.

27. The kit of claim 26 wherein the host cell is an Sf9 cell.

28. A method of detecting endotoxin in a test sample, comprising the steps of:
contacting a test sample with the reagent of claim 1 to form a test sample-reagent mixture; 
contacting the test sample-reagent mixture with a Factor C substrate, wherein cleavage of the Factor C substrate generates a detectable signal to form a test sample-substrate-reagent mixture; and 
assaying the test sample-substrate-reagent mixture for the presence or absence of the detectable signal, wherein an amount of the detectable signal that is increased relative to a control sample that does not contain endotoxin indicates a presence of endotoxin in the test sample.

29. A method of detecting endotoxin in a test sample, comprising the steps of:
contacting a test sample with a reagent comprising (a) a surfactant in an amount which increases sensitivity of an enzymatic endotoxin reaction relative to a reagent containing a purified horseshoe crab Factor C protein but which does not comprise the surfactant, and (b) a recombinant Carcinoscorpius rotundicauda Factor C protein, wherein the Factor C protein is made by the method of culturing a host cell comprising a vector encoding the Factor C protein in a supernatant under conditions such that the Factor C protein is expressed into the supernatant, 

wherein the Factor C protein is enzymatically active in the presence of the surfactant to form a test sample-reagent mixture;
contacting the test sample-reagent mixture with N-t-BOC-Asp(Obzl)-Pro-Arg-7-Amido-4-methyl coumarin to form a test substance-substrate-reagent mixture; and 
assaying the test sample-substrate-reagent mixture for the presence or absence of a fluorescent signal, wherein an amount of the fluorescent signal that is increased relative to a control sample that does not contain endotoxin indicates a presence of endotoxin in the test sample.

30. A reagent for increasing sensitivity of enzymatic endotoxin detection comprising:
a purified recombinant Carcinoscorpius rotundicauda Factor C protein; and ZWITTERGENT 3-14, 

wherein the Carcinoscorpius rotundicauda Factor C protein is enzymatically active in the presence of the ZWITTERGENT 3-14 and wherein the ZWITTERGENT 3-14 is present in an amount that increases the sensitivity of an enzymatic detection reaction relative to a reagent that comprises the purified recombinant Carcinoscorpius rotundicauda Factor C protein but does not comprise the ZWITTERGENT 3-14.

31. The reagent of claim 30 further comprising a Factor C substrate.

32. The reagent of claim 31 wherein the Factor C substrate is N-t-BOC-Asp(Obzl)-Pro-Arg-7-Amido-4-methyl coumarin.

33. The reagent of any one of claims 1 through 9 wherein the surfactant is ZWITTERGENT 3-14.

34. The reagent of claim 1 further comprising a Factor C substrate.

35. The reagent of claim 34 wherein the Factor C substrate is N-t-BOC-Asp(Obzl)-Pro-Arg-7-Amido-4-methyl coumarin.

36. A method of detecting endotoxin in a test sample, comprising the steps of:
contacting a test sample with the reagent of claim 30 to form a test sample-reagent mixture; 
contacting the test sample-reagent mixture with a Factor C substrate, wherein cleavage of the Factor C substrate generates a detectable signal, to form a test sample-substrate-reagent mixture; and 
assaying the test sample-substrate-reagent mixture for the presence or absence of the detectable signal, wherein an amount of the detectable signal that is increased relative to a control sample that does not contain endotoxin indicates a presence of endotoxin in the test sample.

37. A method of detecting endotoxin in a test sample, comprising the steps of:
contacting a test sample with (1) the reagent of claim 30 and (2) N-t-BOC-Asp(Obzl)-Pro-Arg-7-Amido-4-methyl coumarin to form a test sample-substrate-reagent mixture; and 
assaying the test sample-substrate-reagent mixture for the presence or absence of a fluorescent signal, wherein an amount of the fluorescent signal that is increased relative to a control sample that does not contain endotoxin indicates a presence of endotoxin in the test sample.

38. A kit comprising:
the reagent of claim 30; and 
instructions for the method of claim 37.

39. The kit of claim 38, further comprising a Factor C substrate.

40. The kit of claim 39 wherein the Factor C substrate is N-t-BOC-Asp(Obzl)-Pro-Arg-7-Amido-4-methyl coumarin.

41. A reagent for increasing sensitivity of enzymatic endotoxin detection, comprising:
0.03-3 μg/ml of a purified horseshoe crab Factor C protein; and 
a surfactant, 

wherein the purified horseshoe crab Factor C protein is enzymatically active in the presence of the surfactant and wherein the surfactant is present in an amount which increases the sensitivity of an enzymatic endotoxin detection reaction relative to a reagent containing the purified horseshoe crab Factor C protein but which does not comprise the surfactant.

42. A reagent for increasing sensitivity at enzymatic endotoxin detection, comprising:
a purified horseshoe crab Factor C protein; and 
0.001-0.003% of a surfactant, 

wherein the purified horseshoe crab Factor C protein is enzymatically active in the presence of the surfactant and wherein the surfactant is present in an amount which increases the sensitivity of an enzymatic endotoxin detection reaction relative to a reagent containing the purified horseshoe crab Factor C protein but which does not comprise the surfactant.

43. A reagent for increasing sensitivity of enzymatic endotoxin detection, comprising:
0.03-3 μg/ml of a purified recombinant Carcinoscorpius rotundicauda Factor C protein; and 
ZWITTERGENT 3-14, 

wherein the Carcinoscorpius rotundicauda Factor C protein is enzymatically active in the presence of the ZWITTERGENT 3-14 and wherein the ZWITTERGENT 3-14 is present in an amount that increases the sensitivity of an enzymatic detection reaction relative to a reagent that comprises the purified recombinant Carcinoscorpius rotundicauda Factor C protein but does not comprise the ZWITTERGENT 3-14.

44. A reagent for increasing sensitivity of enzymatic endotoxin detection, comprising:
a purified recombinant Carcinoscorpius rotundicauda Factor C protein; and 
0.0025% of ZWITTERGENT 3-14, 

wherein the Carcinoscorpius rotundicauda Factor C protein is enzymatically active in the presence of the ZWITTERGENT 3-14 and wherein the ZWITTERGENT 3-14 is present in an amount that increases the sensitivity of an enzymatic detection reaction relative to a reagent that comprises the purified recombinant Carcinoscorpius rotundicauda Factor C protein but does not comprise the ZWITTERGENT 3-14.